Methods of Detecting Listeria from an Environmental Sample

ABSTRACT

The present invention relates to the identification of microorganisms from an environmental sample, and in particular to the rapid identification of Listeria spp. The methods and kits described herein provide a method of detecting Listeria spp. without the need for an enrichment step.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Application Ser.No. 62/218,678, filed Sep. 15, 2015. The entire contents of theaforementioned application are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to the identification of microorganismsfrom an environmental sample, and in particular to the rapididentification of Listeria spp.

BACKGROUND

Listeria spp. are ubiquitous bacteria widely distributed in theenvironment. Among the species of Listeria, only Listeria monocytogenesis commonly pathogenic for humans. However, some other species ofListeria are pathogenic. Listeria ivanovii, for example, is a pathogenof mammals, specifically ruminants, and while not pathogenic for humans,it is pathogenic for animals.

Listeria is the causative agent of the relatively rare bacterialdisease, listeriosis, which affects primarily pregnant women, newborns,adults with weakened immune systems, and the elderly. Food-bornetransmission constitutes the main route of acquisition of listeriosis.Listeria can be found in soil, which can lead to vegetablecontamination; however, animals can also be carriers.

Listeriosis is a serious disease for humans; the two main clinicalmanifestations are sepsis and meningitis. Meningitis is oftencomplicated by encephalitis, or meningoencephalitis, a pathology that isunusual for bacterial infections. Although human listeriosis occurs onlysporadically, several outbreaks have been observed in recent years.Despite efficient antibiotic therapy, listeriosis represents a publichealth problem due to fatality in up to 30% of cases.

Listeria is an extremely hardy organism that can survive in the cold formany years in naturally contaminated sources. Listeria spp. can be foundthroughout the environment, and therefore, there is a potential forcontamination in a wide variety of foods. Meat, eggs, chicken, seafood,dairy products and vegetables have all been identified as sources ofListeria outbreaks. Food processors, packaging facilities and publichealth officials have recognized Listeria spp. as an indicator ofcontamination of food, water and environmental conditions, thusprocedures exist for monitoring foods, water and the environment forcontamination requiring measurement of Listeria spp. However, rapiddetection and identification of Listeria spp. is essential to the foodindustry and existing methods are dependent on enrichment of the sampleto increase the concentration of target organisms, or other types ofprolonged incubation steps required to increase the concentration oftarget molecules to detectable levels. As such, detection can take up to24-48 hours. Some methods purport not to require an enrichment step;however, the methods still require long incubation periods of up to 6-8hours.

A specific example of such a system is Sample6 DETECT. While theprocedure is said to be “enrichment-free,” it uses a phage to infectspecific bacterial cells and amplify a signal, which requires anincubation of 4-8 hours. Likewise, the DuPont BAX® system is referred toas a “no enrichment” Listeria assay, but requires a “resuscitation” stepof 4 hours.

Accordingly, there is a need for a method and diagnostic tool thatrapidly detects and identifies Listeria spp. in environmental samples.These needs and other needs are satisfied by the devices, methods, andkits of the present invention.

SUMMARY OF THE INVENTION

The purpose of the method is to provide users with a means to assay forthe presence of Listeria spp. in environmental samples from foodproduction, food processing or food service sites without enrichment orincubation prior to performing the diagnostic assay itself. In oneembodiment, the method utilizes the ANSR® Listeria spp. isothermalnucleic acid amplification assay in conjunction with a modified samplelysis procedure. In some embodiments, the entire collected sample issubjected to the lysis procedure, which is conducted in a small volumeof lysis buffer. A benefit of the method is that results can be obtainedin approximately 50 minutes from sample collection, compared to severalhours for methods of lower sensitivity that are dependent on enrichmentof the sample to increase the concentration of target organisms, orother types of prolonged incubation steps required to increase theconcentration of target molecules to detectable levels.

In some embodiments, the invention provides a method for detectingListeria spp. in an environmental sample, the method comprising thesteps of: collecting an environmental sample with a collection device;expressing the collection device in lysis buffer; and performing apathogen diagnostic assay that targets bacterial RNA sequences fordetection and amplification of Listeria RNA in the sample. In someembodiments, the environmental sample is positive for Listeria RNA. Insome embodiments, the environmental sample is negative for Listeria RNA.

In some embodiments, the pathogen diagnostic assay utilizes isothermalamplification of the RNA. In some embodiments, the pathogen diagnosticassay is the ANSR® Listeria spp. isothermal nucleic acid amplificationassay.

In some embodiments, the collection device is a swab, a q-tip or asponge. In some embodiments, the collection device is pre-moistened withletheen broth.

In some embodiments, the time from collection of the sample tocompletion of the pathogen diagnostic assay is 1 hour or less.

In some embodiments, the assay is of sufficient sensitivity to detect aslittle as 1-2 CFU in the environmental sample without enrichment of thesample.

In some embodiments, the volume of lysis buffer is about 1.0 ml. In someembodiments, about 0.5 ml of the sample in lysis buffer is used for thepathogen diagnostic assay.

In some embodiments, the invention provides a kit for detecting thepresence of Listeria spp. in an environmental sample comprising acontainer, the container comprising: a collection device for collectingan environmental sample; lysis buffer for expressing the collectiondevice; and a pathogen diagnostic assay that targets bacterial RNAsequences for detection and amplification of Listeria RNA in the sample.

In some embodiments of the kit, the collection device is a swab. In someembodiments, the collection device is pre-moistened with letheen broth.

In some embodiments of the kit, the container further comprises sampletubes.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention may be better understood when the following detaileddescription is read with reference to the accompanying drawings.

FIG. 1 depicts the key steps in the Reference Method and ExperimentalMethod.

FIG. 2 depicts results for Test Site 1.

FIG. 3 depicts results for Test Site 2.

FIG. 4 depicts results for Test Site 3.

FIG. 5 depicts results for Test Site 4

FIG. 6 depicts results for Test Site 5.

FIG. 7 depicts results for Test Site 6.

FIG. 8 depicts results for Test Site 7.

FIG. 9 depicts composite results for all test sites.

DETAILED DESCRIPTION

In the following description, numerous specific details are given toprovide a thorough understanding of the embodiments. The embodiments canbe practiced without one or more of the specific details, or with othermethods, components, materials, etc. In other instances, well-knownstructures, materials, or operations are not shown or described indetail to avoid obscuring aspects of the embodiments.

Reference throughout this specification to “one embodiment,” “anembodiment,” or “embodiments” means that a particular feature,structure, or characteristic described in connection with the embodimentis included in at least one embodiment. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Unless indicated otherwise, when a range of any type is disclosed orclaimed, it is intended to disclose or claim individually each possiblenumber that such a range could reasonably encompass, including anysub-ranges encompassed therein. Moreover, when a range of values isdisclosed or claimed, which Applicants intend to reflect individuallyeach possible number that such a range could reasonably encompass,Applicants also intend for the disclosure of a range to reflect, and beinterchangeable with, disclosing any and all sub-ranges and combinationsof sub-ranges encompassed therein.

Methods and reagents for isothermal detection and amplification of RNAare described in International Patent Application Publication No. WO2009/012246, which is incorporated by reference herein in its entirety.Briefly, the methods of amplifying nucleic acid target sequences rely onnicking and extension reactions to amplify shorter sequences in ashorter timeframe than traditional amplification reactions, such as, forexample, strand displacement amplification reactions. Embodiments of theinvention include, for example, reactions that use only two templates toamplify a target sequence, one or two nicking enzymes, and a polymerase,under isothermal conditions. In exemplary embodiments, the polymeraseand the nicking enzyme are thermophilic, and the reaction temperature issignificantly below the melting temperature of the hybridized targetregion. The nicking enzyme nicks only one strand in a double-strandedduplex, so that incorporation of modified nucleotides is not necessaryas in the case of conventional strand displacement amplification. Aninitial heat denaturation step is not required for the methods of thepresent invention. Due to the simplicity of the reaction, in exemplaryembodiments, the reaction is very easy to perform, requires no specialequipment, such as a thermocycler, and can amplify 20-30mer products 108to 1010 fold from genomic DNA in only about 2.5 to about 10 minutes. Themethod is able to amplify RNA during a simultaneous reversetranscription step.

The ANSR® Listeria assay is an isothermal detection system that targetsribosomal RNA (rRNA), a high copy number target. Lysis of even a singleListeria cell can release on the order of 1,000 to 10,000 copies ofrRNA. Since the lysis reaction is performed in a small volume, targetconcentration is sufficiently high that a 5-50 uL aliquot of the lysatesubsequently transferred to the ANSR® assay reagent tube will contain asufficient number of target rRNA molecules for detection.

One of ordinary skill in the art would recognize that various otherpathogen diagnostic assays that target bacterial RNA sequences fordetection and amplification of Listeria RNA in the sample may be used inthe invention. In some embodiments, the pathogen diagnostic assay is theAtlas® Listeria LSP Detection Assay (Roka Bioscience). In someembodiments, the pathogen diagnostic assay is the BAX® System Real-TimePCR Assay for Genus Listeria (DuPont).

Providing a sample to be tested may comprise providing a sample that issuspected of containing a target microorganism. The sample can be anysample that may include a target microorganism as defined herein.Nonlimiting examples of suitable samples include environmental samples(e.g., surface swabs/sponges, soil, sediments, fomites), food (e.g., rawmaterials, in-process samples, and finished-product samples), beverages,clinical/veterinary samples (e.g., blood, serum, plasma, urine, sputum,tissue, mucous, feces, wound exudate, pus, cerebrospinal fluid), andwater (e.g., surface water, potable water, process water).

In some embodiments, the presence or absence of a target microorganismcan be analyzed in a test sample that is derived from a variety of food,beverage, or food- or beverage-processing environmental sources.Non-limiting examples of food sources include raw or processed meat, rawor processed fruits or vegetables, non-fluid dairy products (e.g.,cheese, butter, and ice cream), nuts, spices, ingredients, and syrups.Non-limiting examples of beverage sources include potable water, fruitor vegetable juices, milk, and fermented beverages.

Pasteurized food or beverages may also be suitable sources. Non-limitingexamples of food- or beverage-processing environmental samples includefood-handling surface samples (e.g., conveyor belts, blades, cuttingsurfaces, mixing equipment surfaces, filters, storage containers), roomsamples (e.g., walls, floors, drains, ventilation equipment), andcleaning equipment (e.g., hoses, cleaning tools).

In some embodiments, the presence or absence of a target microorganismcan be analyzed in a sample that is derived from a variety of human oranimal sources, such as a physiological fluid, e.g., blood, saliva,ocular lens fluid, synovial fluid, cerebral spinal fluid, pus, sweat,exudate, urine, mucus, lactation milk, or the like. Further, the testsample may be derived from a body site, e.g., wound, skin, nares, scalp,nails, etc.

Samples of particular interest from human or animal sources includemucus-containing samples, such as nasal samples (from, e.g., anterialnares, nasopharyngeal cavity, nasal cavities, anterior nasal vestibule,etc.), as well as samples from the outer ear, middle ear, mouth, rectum,vagina, or other similar tissue. Examples of specific musosal tissuesinclude buccal, gingival, nasal, ocular, tracheal, bronchial,gastrointestinal, rectal, urethral, ureteral, vaginal, cervical, anduterine mucosal membranes.

Besides physiological fluids, other test samples may include otherliquids as well as solid(s) dissolved in a liquid medium. Samples ofinterest may include process streams, water, soil, plants or othervegetation, air, surfaces (e.g., contaminated surfaces), and the like.Samples can also include cultured cells. Samples can also includesamples on or in a device comprising cells, spores, or enzymes (e.g., abiological indicator device).

Suitable samples for methods of the present disclosure can includecertain solid samples. Solid samples may be disintegrated (e.g., byblending, sonication, homogenization) and may be suspended in a liquid(e.g., water, buffer, broth). In some embodiments, a sample-collectiondevice (e.g., a swab, a sponge) containing sample material may be usedin the method.

Alternatively, the sample material may be eluted (e.g., rinsed, scraped,expressed) from the sample-collection device before using the samplematerial in the method. In some embodiments, liquid or solid samples maybe diluted in a liquid (e.g., water, buffer, broth).

The sample may comprise an indicator microorganism, as described herein.The indicator microorganism can be indicative of contamination (e.g.,fecal contamination), infection (e.g., infection with a pathogenicmicroorganism), or an indicator of general sanitation (e.g., any aerobicmicroorganism). The indicator microorganism further can be a targetmicroorganism.

Microorganisms of particular interest, which may be of interest as anindicator organism or a target microorganism, include prokaryotic andeukaryotic organisms, particularly Gram positive bacteria, Gram negativebacteria, fungi, Mycoplasma, and yeast. Particularly relevant organismsinclude members of the family Enterobacteriaceae, or the familyMicrococcaceae or the genera Staphylococcus spp., Streptococcus spp.,Pseudomonas spp., Enterococcus spp., Salmonella spp., Legionella spp.,Shigella spp. Yersinia spp., Enterobacter spp., Escherichia spp.,Bacillus spp., Listeria spp., Vibrio spp., Corynebacteria spp. as wellas, Aspergillus spp., Fusarium spp., and Candida spp. Particularlyvirulent organisms include Staphylococcus aureus ( ), S. epidermidis,Streptococcus pneumoniae, S. agalactiae, S. pyogenes, Enterococcusfaecalis, Bacillus anthracis, Pseudomonas aeruginosa, Escherichia coli,Aspergillus niger, A. fumigatus, A. clavatus, Fusarium solani, F.oxysporum, F. chlamydosporum, Listeria monocytogenes, Listeria ivanovii,Vibrio cholera, V parahemolyticus, Salmonella cholerasuis, S. typhi, S.typhimurium, Candida albicans, C. glabrata, C. krusei, Cronobactersakazakii,

Gram positive and Gram negative bacteria are of particular interest. Ofparticular interest are Gram positive bacteria, such as Listeriamonocytogeness.

Collection Device

Inoculating the collection device can be done by a variety of methodsthat are known in the art. Nonlimiting examples of suitable inoculationmethods include pour-plate techniques, surface inoculation techniques,streak-plating techniques, swab-plating techniques, and surfacecontact-plating techniques (e.g., Rodac plating methods). Filtermembrane plating techniques may be used in the present method, providedthat the membrane filter does not substantially interfere with thereaction between the microorganisms and the indicator systems orinterfere with the observation of the indicator systems.

In some embodiments, the collection device is a swab. The swab may becomprised of cotton or polyester and may be pre-moistened in solution.In some embodiments, the swab is pre-moistened in Letheen broth. Inother embodiments, the swab is pre-moistened in Neutralizing Buffer,Buffered Peptone Water, or culture medium.

In some embodiments, the swab is provided as a polypropylene tube andcap containing a Letheen broth solution and a swab with polypropyleneshaft and polyester fiber tip. In some embodiments, the swab is sold asVeriswab™ Samplers with Letheen Broth (World Bioproducts LLC). In someembodiments, the swab is sold as 3M™ Swab-Sampler with Letheen Broth. Insome embodiments, the swab is sold as 3M™ Quick Swab.

Definitions

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

As used herein, “a,” “an,” “the,” “at least one,” and “one or more” areused interchangeably. Thus, for example, a microorganism can beinterpreted to mean “one or more” microorganisms.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

As used herein, the terms “Listeria” and “Listeria spp.” are usedinterchangeably and refer to all species of the Gram-positive rod shapedbacteria, including, but not limited to, L. grayi, L. innocua, L.ivanovii, L. monocytogenes, L. seeligeri, L. welshimeri.

As used herein, “ANSR® for Listeria” and “ANSR® for Listeriamonocytogenes” refer to an isothermal amplification reaction test methodthat exponentially amplifies the DNA of any bacteria present in samplesto detectable levels in only minutes.

As used herein, “lysis buffer” refers to a liquid buffer containingcomponents to break open bacterial cells and release nucleic acid. Thelytic components may be, but are not limited to, detergents, enzymes ordenaturing salts. In some embodiments, the lysis buffer is the ANSR™Lysis Buffer.

Experimental Method

The Experimental Method is a method of detecting Listeria spp. from anenvironmental sample without the need for an enrichment period. In oneembodiment, the method utilizes the ANSR® Listeria spp. isothermalnucleic acid amplification assay in conjunction with a modified samplelysis procedure. In some embodiments, the entire collected sample issubjected to the lysis procedure, which is conducted in a small volumeof lysis buffer.

In some embodiments, the assay result is read by an automated reader. Inother embodiments, the assay result is detected by enzymatic detectionmethods or gel electrophoresis.

Reference Method

The USDA-MLG protocol for enrichment of Listeria in environmentalsamples was used for culture confirmation. Isolation and Identificationof Listeria monocytogenes from Red Meat, Poultry and Egg Products, andEnvironmental Samples (USDA/FSIS Microbiology Laboratory Guidebook, MLG8.09, Effective May 1, 2013), incorporated by reference herein in itsentirety. FIG. 1 demonstrates the key steps in both the Reference Methodand Experimental Method. The time to completion for the ExperimentalMethod is approximately 50 minutes, as compared to 3-5 days for theReference Method.

EXAMPLES

The following examples are offered by way of illustration, not by way oflimitation.

Example 1: Culture Confirmation Using USDA-MLG Protocol for ListeriaEnrichment in Parallel with Experimental Method

Samples were collected in parallel and analyzed using both theExperimental Method and the Reference Method. A total of 50-100environmental swab samples were collected from a processing plant with agoal of at least 20-25 positives using the following protocol:

Sample Collection

1. Collect environmental sample using swabs that are pre-moistened withletheen broth. Hold two swabs together side-by-side and sample the areato be tested.

2. Keep the paired swab samples together and hold at 2-8° C. untiltesting. Testing should be conducted within 24 hours, preferably thesame day as collection.

Experimental Method Sample Preparation

1. Label the appropriate number of microcentrifuge tubes and place in arack.

2. Prepare ANSR® lysis reagent solution by reconstituting 1 vial oflyophilized lysis reagents with 18 mL of lysis buffer by adding thebuffer to the reagent vial. Swirl gently to mix. a. One vial of lysisreagents is enough for approximately 18 samples. Prepared lysis reagentsolution can be stored at 2-8° C. for 30 days.

3. Add 1 mL of prepared lysis reagent solution to each microcentrifugetube.

4. Place one swab from each pair into the microcentrifuge tube, swirland mix gently with up and down movement in the liquid for severalseconds.

5. Remove swab from the microcentrifuge tube and discard.

6. Cap tubes and proceed to ANSR Test Procedure (Section D).

Experimental Method Test Procedure

1. Preheat the first lysis heater block to 37±2° C. and the second lysisheater block to 80±2° C.

2. Start the ANSR software using the computer connected to the ANSRreader.

3. Enter sample IDs and experiment information. The reader will preheatto 56±1° C.

4. Add 500 μL of the extracted swab sample to separate 1.2 mL clustertubes using 1000 μL pipette tips. a. Use a new pipette tip for eachsample.

5. Incubate the cluster tubes at 37±2° C. for 10 minutes.

6. Then transfer the cluster tubes to the 80±2° C. heater block andincubate for 20 minutes. a. Note: The incubation time for this step canbe extended to a maximum of 60 minutes.

7. At least 3 minutes before the end of the lysis step, preheat thecapped ANSR reaction tubes by placing them in the ANSR reader.

8. At the end of the 20 minute 80° C. lysis, remove and discard the capsfrom the ANSR reaction tubes, which was placed in the ANSR reader.

9. With the cluster tubes still in the 80° C. lysis block, use an8-channel pipette and 100 μL filtered tips, transfer 50 μL of the lysedsample to the designated reaction tubes in the ANSR reader. Place thepermanent caps on the ANSR reaction tubes.

10. After capping, remove the tubes from the reader and vortex briefly(˜2 seconds) then place back into the reader without delay. Close thelid of the ANSR reader.

11. Press START on the ANSR software to begin the assay.

12. Results indicating the presence or absence of Listeria spp. will bedisplayed in 18 minutes. Any tests producing invalid results should berepeated.

Reference Method Primary Enrichment in UVM Broth

Add 225±5 ml (or 225±5 g) of UVM broth to each sample. Hand mixing is anacceptable alternative for stomaching. To hand mix, briefly massage eachsponge to expel the collection broth into the UVM broth. Incubate at30±2° C. for 20-26 h.

Reference Method Secondary Enrichment in FB or MOPS-BLEB and PrimaryEnrichment Plating of UVM

a. Transfer 0.1±0.02 ml of the UVM enrichment to 10±0.5 ml of FB orMOPS-BLEB. As per media preparation instructions, be sure thatappropriate supplements have been added to the FB prior to inoculation.Incubate inoculated FB tubes at 35±2° C. for 26±2 h or inoculatedMOPSBLEB tubes at 35±2° C. for 18-24 h.

b. Streak a MOX plate. Streak a loopful or a drop approximating 0.1 mlof the UVM over the surface of the plate. Alternatively, dip a sterilecotton-tipped applicator or equivalent into the UVM and swab 25-50% ofthe surface of a MOX plate. Use a loop to streak for isolation from theswabbed area onto the remainder of the plate. Incubate the MOX at 35±2°C. for 26±2 h.

Reference Method Examination of UVM-Streaked MOX, Interpretation/Platingof 26-h FB, and Plating of MOPS-BLEB

Examine the UVM-streaked MOX for colonies with morphology typical ofListeria spp. At 26±2 h, suspect colonies are typically small (ca. 1 mm)and are surrounded by a zone of darkening due to esculin hydrolysis.

i. If suspect colonies are present on MOX, transfer suspect colonies toHL agar.

ii. If no suspect colonies are evident, re-incubate the MOX plate for anadditional 26±2 hour.

iii. Proceed to step b below for FB or step c for MOPS-BLEB.

b. After 26±2 h of incubation, examine the FB for the potential presenceof Listeria monocytogenes, by visual examination of the broth fordarkening due to esculin hydrolysis.

i. If any degree of FB darkening is evident, aseptically dispense a dropapproximating 0.1±0.02 ml of FB onto a MOX plate. Swab or streak 25-40%of the surface of the MOX plate with the FB inoculum. Use a loop tostreak for isolation from the initial swab/streak quadrant onto theremainder of the plate. Incubate the MOX plate at 35±2° C. for 26±2 h.

ii. If no FB darkening is evident, re-incubate the FB at 35±2° C. untila total incubation time of 48±2 h has been achieved.

c. After 18-24 h, streak a MOX plate using a loopful of the MOPS-BLEB,or by streaking a drop approximating 0.1 ml, or aseptically dip asterile cotton-tipped applicator or equivalent into the MOPS-BLEB andswab 25-50% of the surface of a MOX plate. Use a loop to streak forisolation from the swabbed area onto the remainder of the plate.Incubate the MOX at 35±2° C. for 26±2 h.

Reference Method Examination of MOX Plates and Interpretation/Plating of48 h FB

a. Examine and select suspect colonies from any MOX agar plate pendinganalysis (i.e. MOX plates streaked from 26±2 h FB, 18-24 h MOPSBLEB,and/or UVM).

b. Re-examine the FB for evidence of darkening after 48±2 h of totalincubation.

i. If any degree of darkening is evident, swab, streak and incubate aMOX plate.

ii. If no darkening of FB is evident and no suspect MOX and/or HLcolonies have been demonstrated, the sample is considered negative forL. monocytogenes.

Reference Method Isolation and Purification Procedures

a. If suspect colonies are present on MOX from any source, use a loop orequivalent sterile device to contact a minimum of 20 (if available)suspect colonies and collectively streak for isolation on one or more HLagar plates. Alternatively, a swipe of suspect growth representing atleast 20 colonies may be used. Incubate the streaked HL at 35±2° C. for22±4 h.

b. After incubation, examine the HL plate(s) against backlight fortranslucent colonies surrounded by a small zone of β-hemolysis.

i. If at least one suspect colony is clearly isolated, proceed toconfirmatory testing (Section 8.6 below). Hold all HL plates containingsuspect colonies (room temperature or refrigeration) until confirmatorytesting is complete.

ii. If suspect colonies or β-hemolytic growth are present on HL but notclearly isolated, re-streak representative suspect colonies/growth ontoone or more fresh HL plates and incubate.

iii. If no suspect isolates are present on HL, pursue follow-up of MOXand/or HL isolates from other branches of analysis (e.g. FB follow-upvs. UVM Primary Enrichment streak follow-up). If no branch of theanalysis produces suspect β-hemolytic colonies on HL, the sample may bereported as negative for L. monocytogenes.

Results for Test Sites 1-7 are shown in FIGS. 2-9. Overall data showed88.1% assay agreement.

Example 2: Surface Inoculation (Plastic), Compared with USDA Method

ANSR vs. USDA Method

Environmental samples were collected and half of the swabs at each levelwere expressed directly into 450 μl of ANSR® lysis buffer, assayedimmediately on ANSR® Listeria. The remaining swabs were added to 10 mlUVM for USDA reference method. Results are shown in tables 1 and 2.

TABLE 1 Surface Inoculation (Plastic), Compared with USDA Method SampleN CFU/square ANSR (+) USDA (+) Unspiked 5 0 5 0 surface DE blank 5 N/A 3* 0 10⁻⁸ 5 6.17E−01  4* 0 10⁻⁷ 5 6.17E+00 5 0 10⁻⁶ 5 6.17E+01 5 0 10⁻⁵5 6.17E+02 5 0 10⁻⁴ 5 6.17E+03 5 1 10⁻³ 5 6.17E+04 5 0 10⁻² 5 6.17E+05 55 Undilute 5 6.17E+07 5 5 *one invalid result DE blank: 50 μl assayeddirectly, no contact with surface or swab

TABLE 2 Surface Inoculation (Ceramic) Compared with USDA Method Sample NCFU/square ANSR (+) USDA (+) DE BLANK 5 N/A 0 0 BPB BLANK 5 N/A  0* 0Unspiked surface 5 0  1* 0 10⁻⁸ 5 2.06E+00 4 0 10⁻⁷ 5 2.06E+01 5 0 10⁻⁶5 2.06E+02 5 0 10⁻⁵ 5 2.06E+03 5 0 10⁻⁴ 5 2.06E+04 5 0 10⁻³ 5 2.06E+05 50 10⁻² 5 2.06E+06 4 5 Undilute 5 2.06E+08  4* 5 *one invalid in set offive replicates BPB BLANK; surface inoculated with BPB and swabbed DEBLANK; 50 μL assayed directly-no contact with surface or swab

Example 3: Surface Swab Samples Using 1 ml Lysis Buffer Verses MOXStreaking Method

Surface was spiked with pure Listeria monocytogenes culture andenvironmental samples were subjected to the Experimental Method orstreaking on MOX followed by ANSR® Listeria. The results are shown intables 3-6.

TABLE 3 Surface Spiked with Pure Culture LESS Plus Sample Lm ANSR IDCFU/area Experimental Method Listeria MOX BPW 0  pos* neg neg neg negneg neg neg Lm-8 ~1 pos pos pos neg pos pos pos neg Lm-7 ~10 pos pos posneg pos pos pos neg Lm-6 ~100 pos pos pos neg pos pos pos neg Lm-5 ~1000pos pos pos pos pos pos pos pos Lm-4 ~10000 pos pos pos pos pos pos pospos Lm-3 ~100000 pos pos pos pos pos pos pos pos

TABLE 4 Surface Spiked with Listeria monocytogenes in Food Matrix samplespiking CFU/ Experimental Method Culture Method # level area ANSRListeria MOX MOX 1 unspiked 0 neg neg neg 2 pos/neg neg neg 3 −5 ~0.05neg neg neg 4 neg neg neg 5 −4 ~0.5 neg neg neg 6 pos neg neg 7 −3 ~5pos neg neg 8 pos neg neg 9 −2 ~49 pos neg neg 10 pos neg neg 11 −1 ~490pos pos pos 12 pos neg pos

TABLE 5 Surface Spiked with Lm in Food Matrix, Treated with Bleachsample spiking CFU/ Before Beach After Bleach # level area ANSR MOX ANSRMOX 1 unspiked 0 neg neg neg neg 2 neg neg neg neg 3 −5 ~0.2 neg neg negneg 4 neg neg neg neg 5 −4 ~2 pos neg pos neg 6 pos neg pos neg 7 −3 ~20pos pos pos neg 8 pos pos neg neg 9 −2 ~200 pos pos pos neg 10 pos pospos neg 11 −1 ~2000 pos pos pos neg 12 pos pos pos neg

TABLE 6 Surface Spiked with Lm in Food Matrix, Treated with SanitizersExperimental Method Culture Method ANSR Listeria MOX sample spiking CFU/no Mandate no Mandate # level area saniziter plus XY-12 SterBacsaniziter plus XY-12 SterBac 1 unspiked 0 neg NA NA NA neg NA NA NA 2neg NA NA NA neg NA NA NA 3 −5 ~0.3 pos neg pos neg neg neg neg neg 4pos neg neg neg neg neg neg neg 5 −4 ~3.3 pos neg pos neg pos neg negneg 6 neg neg neg pos pos neg neg neg 7 −3 ~33 pos pos pos pos pos negneg neg 8 pos pos pos pos pos neg neg neg

What is claimed is: 1.-12. (canceled)
 13. A kit for detecting thepresence of Listeria spp. in an environmental sample comprising acontainer, the container comprising: a collection device for collectingan environmental sample; lysis buffer for expressing the collectiondevice; and a pathogen diagnostic assay that targets bacterial RNAsequences for detection and amplification of Listeria RNA in the sample.14. The kit of claim 13, wherein the collection device is a swab. 15.The kit of claim 13, wherein the collection device is pre-moistened withletheen broth.